Stimulus‐Dependent Dynamics of P53 in Single Cells

Molecular Systems Biology 7; Article number 488; doi:10.1038/msb.2011.20 Citation: Molecular Systems Biology 7:488 & 2011 EMBO and Macmillan Publishers Limited All rights reserved 1744-4292/11 www.molecularsystemsbiology.com REPORT Stimulus-dependent dynamics of p53 in single cells

Eric Batchelor, Alexander Loewer, Caroline Mock and Galit Lahav*

Department of Systems Biology, Harvard Medical School, Boston, MA, USA * Corresponding author. Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. Tel.: þ 1 617 432 5621; Fax: þ 1 617 432 5012; E-mail: [email protected]

Received 30.8.10; accepted 7.3.11

Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input–output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to c-radiation. We further found that while c-triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input-sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus- dependent dynamics of p53, including excitability and input-duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network. Molecular Systems Biology 7: 488; published online 10 May 2011; doi:10.1038/msb.2011.20 Subject Categories: simulation and data analysis; signal transduction Keywords: DNA damage; dynamics; excitability; feedback; live-cell imaging

This is an open-access article distributed under the terms of the Creative Commons Attribution Noncommercial Share Alike 3.0 Unported License, which allows readers to alter, transform, or build upon the article and then distribute the resulting work under the same orsimilar license to this one. The work must be attributed back to the original author and commercial use is not permitted without speciﬁc permission.

Introduction In addition, the p53 response to DSBs was shown to be excitable—a full p53 pulse is triggered by either a sustained or The tumor suppressor protein p53 is induced in response to a transient input (Batchelor et al, 2008; Loewer et al, 2010). many stress signals (Horn and Vousden, 2007) and activates The mechanism generating excitability in the p53 response to various stress-response programs including cell-cycle arrest, DSBs has not yet been determined. senescence, and apoptosis. An important unanswered ques- Here we show that different stresses trigger different tion is how can a single protein orchestrate such a complex temporal profiles of p53. We found that the p53 dynamical response to different inputs? Much attention in this regard has response to UV significantly differs from the response to DSBs, been focused around the hypothesis that different post- and we provide new mechanistic insights about the role of translational modifications of p53 lead to activation of feedback controlling specific features of p53 dynamics, such as different downstream gene programs (Brooks and Gu, 2003; excitability and input-strength dependency. Bode and Dong, 2004). However, another potential level of regulation, which has not been explored as much in this system, is the temporal dynamics of p53. The dynamical behavior of p53 has been extensively Results and discussion characterized in response to one particular form of DNA We first set out to determine whether a distinct form of DNA damage, double-strand breaks (DSBs) caused by g-radiation or damage leads to a series of undamped pulses as was previously the radiomimetic drug neocarzinostatin (NCS) (Shiloh et al, shown in response to DSBs (Lahav et al, 2004; Geva-Zatorsky 1983). DSBs trigger a series of p53 pulses with fixed amplitude et al, 2006; Batchelor et al, 2008). We chose UV light, which is and duration, independent of the damage dose, whereas the known to cross-link consecutive pyrimidine bases leading to number of pulses increases with higher damage (Lahav et al, the exposure of single-stranded DNA (ssDNA). Previous 2004; Geva-Zatorsky et al, 2006). It is not clear whether other studies showed that population-averaged analysis can mask types of DNA damage lead to similar dynamical behavior. true dynamical responses (Lahav et al, 2004; Tay et al, 2010);

& 2011 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2011 1 Stimulus-dependent dynamics of p53 in single cells E Batchelor et al

A B 100 ng/ml NCS 2 J/m2 UV

800 800 400 400 0 0 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 p53-Venus (AU) p53-Venus (AU) Time (h) Time (h) Time (h) Time (h) Time (h) Time (h)

D C 400 ng/ml NCS 8 J/m2 UV

800 800 400 400 0 0 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 p53-Venus (AU) p53-Venus (AU) Time (h) Time (h) Time (h) Time (h) Time (h) Time (h)

E F 2 2.2 100 ng/ml NCS 2 J/m UV First pulse 2.2 2 200 ng/ml NCS 4 J/m UV 6 J/m2UV 400 ng/ml NCS 1.8 1.8 8 J/m2UV 10 J/m2UV 1.4 1.4

1 1

Average of p53-Venus (AU) 0.6 Average of p53-Venus (AU) 0.6

012345 0 5 10 15 20 25 30 Time (h) Time (h)

G 600 H 400 600 500 400 500 300 400 300 400 300 200 200 300 200 200 100 100 Duration (min) 100

Duration (min) 100 Amplitude (AU) 0 0 Amplitude (AU) 0 0 100 200 400 100 200 400 24 6810 246810 NCS (ng/ml) NCS (ng/ml) UV (J/m2) UV (J/m2) Figure 1 Stimulus-dependent dynamics of p53 in response to NCS (DSBs) and to UV. (A–D) Representative traces of MCF7 cells expressing p53-Venus exposed to 100 (A) or 400 ng/ml (C) of NCS or to 2 (B) or 8 J/m2 (D) UV at the indicated time following treatment. (E, F) p53-Venus levels averaged over the traces of cells in response to various levels of NCS and UV. Note that for NCS, the averaged response is shown only for the first p53 pulse, as synchrony between cells is lost in subsequent pulses. Each experiment includes between 60 and 110 cells. The mean trace was normalized to the mean p53-Venus of the first time point. (G, H) Quantification of the relative amplitude and full-width half-maximum duration of all p53 pulses in response to NCS (G) or the first pulse in response to UV (H). Error bars represent s.e.m.

we therefore analyzed p53 dynamics in single cells using a pulses in response to UV might be independent of the extrinsic p53-Venus fusion (Batchelor et al, 2008). We found that in UV damage, and likely represent pulses resulting from intrinsic response to a single short burst of 2 J/m2 UV, p53 showed damage during normal proliferation as was recently shown pulses that were similar in amplitude and duration to the (Loewer et al, 2010). pulses observed in response to DSBs caused by NCS (Figure 1A The amplitude, duration, and frequency of individual p53 and B). However, the frequency of the pulses in response to UV pulses in response to DSBs are ﬁxed and do not depend on the was lower and did not appear to be as regular as the pulses in damage dose (Lahav et al, 2004; Geva-Zatorsky et al, 2006; response to DSBs. Figure 1A, C, E, and G). To determine whether this is also the To determine whether there was a principal frequency of the case in response to UV, we irradiated cells with a broad range UV-induced pulses, we performed pitch detection as was of UV doses, each delivered in a single burst. We found that previously described (Geva-Zatorsky et al, 2006). In contrast higher UV doses increased both the amplitude and the to the DSB response that results in a principal period of 4–7 h duration of the p53 response (Figure 1B, D, F, and H). No (Geva-Zatorsky et al, 2006), a broad distribution of pitches was principal frequency was identiﬁed by pitch detection for any of determined for the UV response (Supplementary Figure S1), the UV doses (Supplementary Figure S1), indicating that there indicating that there was no dominant frequency of p53 pulses. is no periodicity in the p53 response to UV. This dose- The irregularity of the pulse frequency suggests that later dependent, single p53 pulse in response to UV is in stark

2 Molecular Systems Biology 2011 & 2011 EMBO and Macmillan Publishers Limited Stimulus-dependent dynamics of p53 in single cells E Batchelor et al

A DNA DSBs B ssDNA (γ, NCS) (UV)

ATM ATR

p53 p53

Wip1 Mdm2 Wip1 Mdm2

C D Simulated 2.2 First pulse 2.2 UV dose 1.8 1.8 2 J/m2 UV 4 J/m2 UV 1.4 1.4 6 J/m2 UV 8 J/m2 UV

Simulated Simulated 1 1 10 J/m2 UV

p53 levels (AU) p53 levels (AU) 0.6 0.6 012 3450 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (h) Time (h) Time (h)

EF10 Gy γ 2 J/m2 UV 8 J/m2 UV Time (h) 012345678910 Time (h) 01234560123456 Chk2-P(T68) Chk1-P(S317) (ATM activity) (ATR activity) Chk2 Chk1

Tubulin Tubulin

G 10 Gy γ 8 J/m2 UV H 10 Gy γ 8 J/m2 UV Time (h) 0 0.5 1 2 3 4 0 0.5 1 2 3 4 Time (h) 0 0.5 1 2 3 4 0 0.5 1 2 3 4 Chk2-P(T68) Chk1-P(S317) (ATM activity) (ATR activity)

Chk2 Chk1

Tubulin Tubulin

1.5 1.5 10 Gy γ 10 Gy γ 2 8 J/m2 UV 1 8 J/m UV 1 Chk2-P 0.5 Chk1-P 0.5 Normalized Normalized 0 0 04132 04132 Time (h) Time (h) Figure 2 A single feedback switches repeated, fixed p53 pulses into a single dose-dependent pulse. (A, B) Diagrams showing key species of the p53 signaling networks in response to DSBs (A) and UV (B). Dashed lines indicate transcription; solid lines indicate protein–protein interactions. (C, D) Simulated p53 levels in response to DSBs (C) and UV (D). See Supplementary information for a description of the model. (E–H) Western blot analysis of ATM activity (measured by Chk2-P) and ATR activity (measured by Chk1-P) in cells irradiated with g or UV. Quantification of Chk1-P and Chk2-P levels were normalized to tubulin levels. Error bars represent s.e.m. of triplicate experiments. contrast to the repeated fixed pulses in response to DSBs responding to DSBs or to UV. In both networks, PI3 kinase- (Figure 1; Supplementary Movies 1 and 2). related kinases (ATM or ATR) relay the damage signal to p53, How can a single protein respond to different inputs with activating two core negative-feedback loops (Figure 2A and B). such markedly different dynamics? To address this question, One feedback loop is between p53 and the E3 ubiquitin ligase we searched for differences in the architecture of the networks Mdm2, in which p53 activates Mdm2 expression and Mdm2

& 2011 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2011 3 Stimulus-dependent dynamics of p53 in single cells E Batchelor et al

A 400 ng/ml NCS B 8 J/m2 UV Control +Wm Control +Wm 1200 1200 300 300 800 200 200 800 100 100 400 400 p53-Venus (AU) p53-Venus (AU) p53-Venus (AU) p53-Venus (AU) 0 0 0 0 061 2 3 4 5 061 2 3 4 5 061 2 3 4 5 061 2 3 4 5 Time (h) Time (h) Time (h) Time (h)

C 0.6 D 0.6 α α 0.5 Control 0.5 Control +Wm +Wm 0.4 β 0.4 β 0.3 0.3 0.2 0.2 0.1 0.1 Fraction of cells Fraction of cells 0 0 041 2 3 0 1 2 3 4 α/β α/β

EFDNA DSBs Inhibition + ssDNA Inhibition γ ATM Mdm2 ATR Mdm2 ( , NCS) Rapid (UV) Ser-395 Ser-407 degradation P P G H 10 Gy γ 10 Gy γ 8 J/m2 UV Plasmid pMdm2 pMdm2S395A Time (h) 020.51 00.5 1 2 Time (h) 020.51 00.5 1 2 Mdm2 Mdm2

Tubulin Tubulin

2.5 2 2 1.5 1.5 1 1 Mdm2 10 Gy γ 0.5 pMdm2

Normalized 0.5 2

8 J/m UV Normalized pMdm2S395A 0 0 0 0.51 1.5 2 0 0.51 1.5 2 Time (h) Mdm2 + Mdm2S395A Time (h)

I 400 ng/ml NCS Control +Wm pmCherry + pmCherry + Plasmid pmCherry pmCherry pMdm2S395A pMdm2S395A 60 60 60 60

40 40 40 40 20 20 20 20 p53-Venus (AU) p53-Venus (AU) p53-Venus (AU) 0 0 0 p53-Venus (AU) 0 06123 45 06123 45 06123 45 06123 45 Time (h) Time (h) Time (h) Time (h) J 100 pmCherry 80 pmCherry + pMdm2S395A 60 40 20 % of cells with % of cells

a p53-Venus pulse a p53-Venus 0 Control + Wm

4 Molecular Systems Biology 2011 & 2011 EMBO and Macmillan Publishers Limited Stimulus-dependent dynamics of p53 in single cells E Batchelor et al

targets p53 for degradation (Barak et al, 1993; Wu et al, 1993; minimal effect on p53 dynamics compared with simulations in Haupt et al, 1997; Kubbutat et al, 1997). The other feedback is which cross-talk was absent (Supplementary Figure S3; between p53 and the phosphatase Wip1, in which p53 Supplementary information). We concluded that the low level activates Wip1 expression and Wip1 dephosphorylates p53, of cross-talk between the DSB and UV pathways does not alter thus reducing its stability (Fiscella et al, 1997; Lu et al, 2005). the dynamical behavior of p53. However, we noted one main difference: the network We next asked whether the p53 response to UV exhibits responding to DSBs includes a negative feedback between excitable behavior as was shown in response to DSBs p53 and ATM mediated by Wip1 (Shreeram et al, 2006; (Batchelor et al, 2008; Loewer et al, 2010; Figure 3A and C). Figure 2A, red inhibitory arrow). In contrast, as Wip1 acts A hallmark of excitability is that a transient input is sufﬁcient through dephosphorylation, and ATR does not require for triggering a full response. We generated a transient input phosphorylation for its activity (Cimprich and Cortez, 2008), signal by challenging cells with UVand inhibiting ATR 1 h post- we assumed no direct inhibition of ATR by Wip1 (Figure 2B). damage, using the PI3-kinase-like kinase inhibitor wortman- To determine the contribution of the Wip1-ATM feedback to nin (Sarkaria et al, 1998; Supplementary Figure S4). We found p53 dynamics, we used our mathematical model of p53 pulses that in response to UV,p53 immediately stopped accumulating in response to DSBs (Batchelor et al, 2008; Figure 2C) and when the upstream damage signal was inhibited (Figure 3B removed the negative feedback from Wip1 to ATM (Supple- and D; Supplementary Figure S5). These results show that the mentary information). This perturbation was sufﬁcient p53 response to UV, as opposed to the response to DSBs, is not for switching p53 dynamics from repeated pulses into a single excitable. Wortmannin is a broad-range inhibitor and we pulse (Supplementary Figure S2). In addition, our model therefore cannot exclude the possibility that the p53 pulse is predicts that, in contrast to the pulsatile dynamics of ATM in halted because of the inhibition of other PI3-kinase-like response to DSBs (Batchelor et al, 2008; Figure 2E), active ATR kinases. However, as ATR is the kinase dominating the will show a single continuous pulse with a duration that response to UV, and we show a complete inhibition of ATR depends on the level of UV (Supplementary Figure S2). activity in the presence of wortmannin (Supplementary Figure Western blot analysis of ATR activity in response to low and S4), lack of excitability in response to UV supports the high UV was consistent with this prediction (Figure 2F). To suggestion that the p53 response depends on continuous computationally recapitulate the dose-dependent amplitude activation of ATR. of p53 pulses observed experimentally in response to UV We next sought to identify possible mechanisms that could (Figure 1F), we were required to vary the production rate of generate excitability in response to DSBs but not in response to active ATR in a dose-dependent manner (Supplementary UV. One mechanism for generating excitability is to have a fast information; Figure 2D). Indeed, we found experimentally positive feedback (Hodgkin and Huxley, 1952; Suel et al, that the levels of ATR substrate, Chk1-P, depended on the UV 2006). The currently known positive feedbacks in the p53 dose (Figure 2F). Although these data cannot rule out the response to DSBs function on a transcriptional timescale possibility that Chk1-P levels might be regulated by an ATR- (Harris and Levine, 2005), and therefore are not likely to act independent phosphatase, our results are consistent with a quickly enough to generate the observed behavior. Another dose-dependent ATR activity. In agreement, in our previous possible mechanism for generating excitable behavior is fast work we showed that modulation of the DSB network by Wip1 removal of an inhibitor, (Suel et al, 2006; Howlett et al, 2008), knockdown resembled the UV response, generating a dose- such as Mdm2. Indeed, Stommel and Wahl (2004) showed that dependent p53 response to DSBs (Batchelor et al, 2008). Mdm2 is rapidly degraded in response to NCS (Stommel and Our model and network diagrams (Figure 2A and B) display Wahl, 2004). In our previous work, we showed computation- a complete separation between the DSBs/ATM and the ally that ATM-mediated degradation of Mdm2 contributes to UV/ATR pathways. However, previous studies have shown the accumulation of p53 in response to DSBs (Batchelor et al, cross-talk between the two pathways (Jazayeri et al, 2006; 2008). It is possible that Mdm2 is not rapidly degraded in the Yajima et al, 2009). We therefore sought to determine the early response to UV, preventing p53 excitability. Indeed, we extent of cross-talk in our system and its potential impact on found that Mdm2 levels slowly decrease in response to UV p53 dynamics. We measured the levels of Chk2-P (ATM target) compared with a rapid, relatively large degradation in and the levels of Chk1-P (ATR target) in response to g and UV. response to DSBs (Figure 3G). Our results show relatively low levels of cross-talk between the The differential regulation of Mdm2 in response to DSBs and pathways (Figure 2G and H; Supplementary information). We UV can be explained by the differential phosphorylation of simulated the experimentally measured cross-talk and found Mdm2 in response to these stresses. In response to DSBs,

Figure 3 Differential p53 excitability between DSBs and UV results from differential regulation of Mdm2. (A–D) Cells expressing p53-Venus were treated with NCS (A)orUV(B). One hour after damage, medium containing DMSO (control) or wortmannin ( þ Wm) (Sarkaria et al, 1998) was added (dashed line). Representative single-cell traces of average p53-Venus intensity are shown for each condition. (C) Histogram of the ratio of peak p53-Venus intensity, a, to p53-Venus intensity at time of DMSO or Wm addition, b, for the experiment shown in (A)(4100 cells/condition). (D) Histogram of the ratio of p53-Venus intensity at 6 h after UV, a, to p53-Venus intensity at time of DMSO or Wm addition, b, for the experiment shown in (B)(480 cells/condition). (E, F) Diagrams showing the differences in the negative regulation of Mdm2 by ATM (E) and ATR (F). (G) Western blot analysis and quantiﬁcation of Mdm2 levels in cells irradiated with g or UV. Error bars represent s.e.m. of triplicate experiments. (H) Western blot analysis and quantiﬁcation of WT and mutant Mdm2 levels following g treatment. Note that bands include both endogenous and exogenous Mdm2 or Mdm2S395A. Error bars represent s.e.m. of triplicate experiments. (I) Representative traces of average p53-Venus intensity for cells transfected with control or mutant Mdm2 plasmid in response to NCS. DMSO (control) or wortmannin ( þ Wm) was added 30 min after NCS treatment (dashed line). (J) The percentage of cells that show a p53-Venus pulse for the experiment in (I) (60–100 cells/condition). Error bars represent s.e.m.

& 2011 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2011 5 Stimulus-dependent dynamics of p53 in single cells E Batchelor et al

Mdm2 is phosphorylated by ATM on Ser-395 (Khosravi et al, has already been shown in other organisms including bacteria 1999; Maya et al, 2001; Michael and Oren, 2003). This (Suel et al, 2006) and yeast (Hao et al, 2008), and in other phosphorylation destabilizes Mdm2 by increasing its auto- systems in mammalian cells. For example, stimulation of ubiquitination and subsequent proteasomal degradation PC-12 cells with epidermal growth factor was found to (Stommel and Wahl, 2004) (Figure 3E). In contrast, UV generate a transient Erk kinase activity, resulting in cellular radiation leads to phosphorylation on residue Ser-407 (Shino- proliferation. In contrast, stimulation with neuronal growth zaki et al, 2003), which inhibits Mdm2 activity (Figure 3F). factor led to sustained Erk activity, resulting in differentiation UV therefore reduced p53 access by Mdm2 through a post- (Marshall, 1995; Santos et al, 2007). Further quantitative translational modification, which is rapidly reversible; in investigations of p53 dynamics and cellular outcomes in contrast, DSBs cause degradation of Mdm2, which takes response to various sources of stress are required to determine longer to reverse as mRNA and protein synthesis operate on a whether distinct p53 dynamics are decoded differently by slower timescale than dephosphorylation (hours compared various downstream programs, which will provide new with minutes). opportunities for controlling individual cell fate decisions. To determine whether the rapid degradation of Mdm2 in response to DSBs contributes to the excitability of p53, we mutated Ser-395 of Mdm2 to alanine and monitored Mdm2 Materials and methods and p53 dynamics. The alanine substitution prevented the degradation of Mdm2 in the initial response to DNA DSBs Plasmids and cells (Figure 3H). Transient transfection of the mutated Mdm2 We maintained human breast cancer epithelial MCF7 cells at 371Cin plasmid revealed that this form of Mdm2, even in the presence RPMI supplemented with 10% fetal calf serum, 100 U/ml penicillin, of endogenous Mdm2, reduced the percentage of cells that 100 mg/ml streptomycin, and 250 ng/ml fungizone (Gemini Bio- Products). MCF7-p53-Venus (Batchelor et al, 2008) was grown in showed an excitable p53 pulse (Figure 3I and J) in response to medium supplemented with 400 mg/ml of G418. For UV-irradiated DSBs. These results suggest that the rapid degradation of cells, we used RPMI lacking riboflavin and phenol red (transparent Mdm2, mediated by ATM in response to DSBs, contributes to medium). For experiments involving wortmannin, we replaced the the excitable behavior of p53. In contrast, lack of rapid Mdm2 medium with fresh medium containing 100 mM wortmannin every degradation in the initial UV response results in non-excitable hour because of the instability of this compound in solution. To generate the plasmid pMdm2, we used MultiSite-Gateway activation of p53. recombination (Invitrogen). We amplified an B3.6-kb portion of the In summary, we showed how p53, a single transcription Mdm2 promoter, the Mdm2 cDNA, and a polyA tail individually by factor responding to multiple stresses in human cells, can PCR with primers containing attB sites, and cloned each into pDONR transmit distinct signals by eliciting different dynamical plasmids by recombination (BP clonase, Invitrogen). We then generated a fusion construct by three-fragment recombination (LR patterns; DSBs lead to a series of excitable pulses, whereas Clonase Plus, Invitrogen) using the pDONR plasmids and a modified UV leads to a graded, non-excitable single pulse. Comparison pDESTR4R3 vector containing a puromycin selection marker. To of the networks responding to these types of damage allowed generate pMdm2S395A, we first used site-directed mutagenesis us to identify and validate, both experimentally and compu- (Stratagene) to convert the TCT codon 395 to a GCT codon in the tationally, the molecular mechanisms responsible for specific Mdm2 cDNA donor vector. We then used three-fragment recombination with the donor vectors for the Mdm2 promoter and the polyA tail. features of p53 dynamics; activation of the core p53–Mdm2- We transfected plasmid pMdm2S395A into MCF7 cells expressing p53- feedback loop by ATR generates a graded single pulse, the Venus using FuGene6 transfection reagent (Roche). Westerns were feedback from Wip1 to ATM is required for repeated fixed performed 48 h after transfection. In microscopy experiments, we pulses, and the rapid degradation of Mdm2 contributes to the identified cells that received the plasmid by co-transfecting an additional plasmid that constitutively expresses mCherry from the excitable behavior of p53. CMV promoter (pmCherry). pmCherry was constructed by inserting It is intriguing to ask why excitability evolved in response to the sequence encoding the red fluorescent protein mCherry into the C1 DSBs but not UV. One potential explanation is that DSBs are expression vector (Clontech). Cells were imaged 3 days after more harmful to cells than UV-induced DNA lesions, as they transfection as described below in time-lapse microscopy. Only cells can give rise to gross chromosomal aberrations. It makes sense expressing mCherry were analyzed. DNA damage was induced in cells using a cobalt-60 g source, NCS that the system would be poised to respond to DSBs by (Sigma), or a UV-C 254 nm light source (Ushio). g-Irradiation was triggering a p53 pulse as a precaution (Loewer et al, 2010). In given in a single burst lasting o4 min. NCS has been shown to act addition, the frequency of ATR-activating stress (e.g. ssDNA) solely within 5 min following addition of the drug to the cell culture during normal growth is much greater than the frequency of medium (Shiloh et al, 1983). The timescale of NCS treatment was therefore comparable to that of g-irradiation. UV was delivered to cells DSBs. It may be detrimental to excite a full p53 response every using a UV lamp with a rate of 1.5 J/m2/s. All UV treatments, therefore, time ssDNA is detected, unnecessarily activating cell-cycle were performed in a single burst lasting o7s. arrest or apoptosis. Our study also raises the question of whether the dynamical response of p53 to other types of stress (such as hypoxia, Immunoblots ribosomal stress, and oncogenic activation) correspond to one We harvested cells and obtained protein samples by lysis, quantifying of the identified classes of dynamics (excitable pulses or a the total protein concentration by Bradford assay. We separated equal graded response), or whether new dynamical behaviors can be amounts of total protein by electrophoresis on 4–12% Bis-Tris gradient identified. Moreover, as DSBs and UV are known to activate gels (Invitrogen). We detected p53 using DO-1 monoclonal antibody et al (mAb; Santa Cruz Biotechnology), Mdm2 using SMP14 mAb (Santa distinct targets of p53 (Zhao , 2000), it is tempting to Cruz Biotechnology), phospho-Chk1(S317) using a rabbit polyclonal speculate that the stimulus-dependent dynamics of p53 antibody (Cell Signaling Technology), phospho-ATM(S1981) using a contribute to this differential regulation. Such a connection mAb (Rockland), b-tubulin using E7 mAb (Developmental Studies

6 Molecular Systems Biology 2011 & 2011 EMBO and Macmillan Publishers Limited Stimulus-dependent dynamics of p53 in single cells E Batchelor et al

Hybridoma Bank), and actin using AC-74 mAb (Sigma). We quantified Geva-Zatorsky N, Rosenfeld N, Itzkovitz S, Milo R, Sigal A, Dekel E, blot images using ImageJ (NIH). Yarnitzky T, Liron Y, Polak P, Lahav G, Alon U (2006) Oscilla- tions and variability in the p53 system. Mol Syst Biol 2: 2006.0033 Time-lapse microscopy Hao N, Nayak S, Behar M, Shanks RH, Nagiec MJ, Errede B, Hasty J, Elston TC, Dohlman HG (2008) Regulation of cell signaling Two days before microscopy, we grew cells in poly-D-lysine-coated glass-bottom plates (MatTek Corporation) in transparent medium dynamics by the protein kinase-scaffold Ste5. Mol Cell 30: 649–656 supplemented with 5% fetal calf serum, 100 U/ml penicillin, 100 mg/ml Harris SL, Levine AJ (2005) The p53 pathway: positive and negative streptomycin, and 250 ng/ml fungizone (Gemini Bio-Products). We feedback loops. Oncogene 24: 2899–2908 imaged cells with a Nikon Eclipse Ti-inverted fluorescence microscope Haupt Y, Maya R, Kazaz A, Oren M (1997) Mdm2 promotes the rapid (Nikon) on which the stage was surrounded by an enclosure to degradation of p53. Nature 387: 296–299 maintain constant temperature, CO2 concentration, and humidity. Hodgkin AL, Huxley AF (1952) A quantitative description of Images of cells in all experiments were taken every 20 min. The Venus membrane current and its application to conduction and filter set was 500/20 nm excitation, 515 nm dichroic beam splitter, and excitation in nerve. J Physiol 117: 500–544 520 nm emission (Chroma). The mCherry filter set was 545/20–25 nm Horn HF, Vousden KH (2007) Coping with stress: multiple ways to excitation, 570 nm dichroic beam splitter, and 600/50–25 nm emission activate p53. Oncogene 26: 1306–1316 (Chroma). We analyzed images using MetaMorph software (Molecular Howlett E, Lin CC, Lavery W, Stern M (2008) A PI3-kinase-mediated Devices) and custom-written Matlab software (Mathworks). We used a negative feedback regulates neuronal excitability. PLoS Genet 4: semi-automated method in which cells are segmented manually using e1000277 brightfield images, followed by automated analysis of the fluorescence Jazayeri A, Falck J, Lukas C, Bartek J, Smith GC, Lukas J, Jackson SP et al images using custom Matlab software (Batchelor , 2008; Loewer (2006) ATM- and cell cycle-dependent regulation of ATR in et al, 2010). Background-subtracted fluorescence levels were used response to DNA double-strand breaks. Nat Cell Biol 8: 37–45 when reporting traces of p53-Venus intensity. Fluorescence levels without background subtraction were used when reporting histograms Khosravi R, Maya R, Gottlieb T, Oren M, Shiloh Y, Shkedy D (1999) of the ratios of fluorescence levels. Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage. Proc Natl Acad Sci USA 96: 14973–14977 Kubbutat MH, Jones SN, Vousden KH (1997) Regulation of p53 Supplementary information stability by Mdm2. Nature 387: 299–303 Supplementary information is available at the Molecular Systems Lahav G, Rosenfeld N, Sigal A, Geva-Zatorsky N, Levine AJ, Elowitz Biology website (www.nature.com/msb). MB, Alon U (2004) Dynamics of the p53-Mdm2 feedback loop in individual cells. Nat Genet 36: 147–150 Loewer A, Batchelor E, Gaglia G, Lahav G (2010) Basal dynamics of p53 reveal transcriptionally attenuated pulses in cycling cells. Cell 142: Acknowledgements 89–100 We thank U Alon, R Kishony, M Springer, J Toettcher, J Purvis, Lu X, Nannenga B, Donehower LA (2005) PPM1D dephosphorylates A Puszynska, all members of our laboratory for helpful discussions; Chk1 and p53 and abrogates cell cycle checkpoints. Genes Dev 19: J Waters and the Nikon Imaging Center at Harvard Medical School for 1162–1174 advice on live-cell imaging. This research was supported by National Marshall CJ (1995) Specificity of receptor tyrosine kinase signaling: Institutes of Health grant GM083303. EB was supported by the transient versus sustained extracellular signal-regulated kinase American Cancer Society, Pamela and Edward Taft Postdoctoral activation. Cell 80: 179–185 Fellowship. AL was supported by fellowships from the German Maya R, Balass M, Kim ST, Shkedy D, Leal JF, Shifman O, Moas M, Research Foundation and the Charles A King Trust. Buschmann T, Ronai Z, Shiloh Y, Kastan MB, Katzir E, Oren M (2001) ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage. Genes Dev 15: 1067–1077 Conflict of interest Michael D, Oren M (2003) The p53-Mdm2 module and the ubiquitin system. Semin Cancer Biol 13: 49–58 The authors declare that they have no conflict of interest. Santos SD, Verveer PJ, Bastiaens PI (2007) Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate. Nat Cell Biol 9: 324–330 Sarkaria JN, Tibbetts RS, Busby EC, Kennedy AP, Hill DE, Abraham RT References (1998) Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res 58: 4375–4382 Barak Y, Juven T, Haffner R, Oren M (1993) mdm2 expression is Shiloh Y, van der Schans GP, Lohman PH, Becker Y (1983) Induction induced by wild type p53 activity. EMBO J 12: 461–468 and repair of DNA damage in normal and ataxia-telangiectasia Batchelor E, Mock CS, Bhan I, Loewer A, Lahav G (2008) Recurrent skin fibroblasts treated with neocarzinostatin. Carcinogenesis 4: initiation: a mechanism for triggering p53 pulses in response to 917–921 DNA damage. Mol Cell 30: 277–289 Shinozaki T, Nota A, Taya Y, Okamoto K (2003) Functional role of Bode AM, Dong Z (2004) Post-translational modification of p53 in Mdm2 phosphorylation by ATR in attenuation of p53 nuclear tumorigenesis. Nat Rev Cancer 4: 793–805 export. Oncogene 22: 8870–8880 Brooks CL, Gu W (2003) Ubiquitination, phosphorylation and Shreeram S, Demidov ON, Hee WK, Yamaguchi H, Onishi N, Kek C, acetylation: the molecular basis for p53 regulation. Curr Opin Cell Timofeev ON, Dudgeon C, Fornace AJ, Anderson CW, Minami Y, Biol 15: 164–171 Appella E, Bulavin DV (2006) Wip1 phosphatase modulates ATM- Cimprich KA, Cortez D (2008) ATR: an essential regulator of genome dependent signaling pathways. Mol Cell 23: 757–764 integrity. Nat Rev Mol Cell Biol 9: 616–627 Stommel JM, Wahl GM (2004) Accelerated MDM2 auto-degradation Fiscella M, Zhang H, Fan S, Sakaguchi K, Shen S, Mercer WE, Vande induced by DNA-damage kinases is required for p53 activation. Woude GF, O’Connor PM, Appella E (1997) Wip1, a novel human EMBO J 23: 1547–1556 protein phosphatase that is induced in response to ionizing Suel GM, Garcia-Ojalvo J, Liberman LM, Elowitz MB (2006) An radiation in a p53-dependent manner. Proc Natl Acad Sci USA 94: excitable gene regulatory circuit induces transient cellular 6048–6053 differentiation. Nature 440: 545–550

& 2011 EMBO and Macmillan Publishers Limited Molecular Systems Biology 2011 7 Stimulus-dependent dynamics of p53 in single cells E Batchelor et al

Tay S, Hughey JJ, Lee TK, Lipniacki T, Quake SR, Covert MW expression patterns using oligonucleotide arrays. Genes Dev 14: (2010) Single-cell NF-kappaB dynamics reveal digital activa- 981–993 tion and analogue information processing. Nature 466: 267–271 Wu X, Bayle JH, Olson D, Levine AJ (1993) The p53-mdm-2 autoregulatory feedback loop. Genes Dev 7: 1126–1132 Molecular Systems Biology is an open-access journal Yajima H, Lee KJ, Zhang S, Kobayashi J, Chen BP (2009) DNA double- published by European Molecular Biology Organiza- strand break formation upon UV-induced replication stress activates ATM and DNA-PKcs kinases. J Mol Biol 385: 800–810 tion and Nature Publishing Group. This work is licensed under a Zhao R, Gish K, Murphy M, Yin Y, Notterman D, Hoffman WH, Tom E, Creative Commons Attribution-Noncommercial-Share Alike 3.0 Mack DH, Levine AJ (2000) Analysis of p53-regulated gene Unported License.

8 Molecular Systems Biology 2011 & 2011 EMBO and Macmillan Publishers Limited